Stem cells are the source of tissue regeneration. Stem cells, depending on the order they appear along the process of ontogeny, can be divided into embryonic stem cells (ESCs) and adult stem cells (ASCs). Depending on the differences in differentiation potential, they can also be divided into totipotent stem cells, pluripotent stem cells, multipotent stem cells and unipotent stem cells. ASCs, depending on their histogenesis, can also be divided into hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, muscle stem cells, and the like. Some transcription factors can be introduced into animal or human somatic cells with gene transfection techniques, allowing the induced somatic cells to be reconstituted into ESC-like pluripotent stem cells, which are referred as induced pluripotent stem cells (iPSCs) [1,2]. ESCs, ASCs, and iPSCs exhibit different advantages and limitations in clinical applications, such as stages of development, origins and how they were collected, and the like. ESCs possess pluripotency differentiation; however, ethical issues, immunologic rejection, and tumorigenicity severely hinder the clinical research and application thereof. iPSCs possess a similar capacity for differentiation with that of ESCs, iPSCs still have tumorigenicity, the efficiency of generating iPSCs by inducing adult cells is extremely low, and the resulting iPSCs through induction has higher canceration rate. These factors greatly decrease safety in clinical applications. The sources of ASCs are very broad and ASCs neither exhibit tumorigenicity nor present ethical issues. It is traditionally considered that ASCs belong to pluripotent stem cells or unipotent stem cells. In recent years, experimental evidence show that ASCs have “plasticity” and not only can differentiate into specific types of cells in a specific pedigree, but also have the capability of differentiating into other pedigrees, which are irrelative in development. This suggests that ASCs have more potential of differentiation than people previously imagined [3-5].
So far, ASCs do not possess unified phenotypes, culture conditions and methods for identification due to the numerous sources. ASCs derived from various tissues show different potential for differentiation. These factors make the studies of ASCs complicated and confusing. It is difficult to establish a relatively homogeneous cell line, resulting [in further difficulties in clinical applications.
Epigenetic modifications generally include DNA methylation, histone modification and RNA modification; while histone modification includes methylation, acetylation, phosphorylation and ubiquitination with the modification sites usually located at the N-terminal of histone. These modifications can change the state of chromatin and even affect the binding of transcription factors to a DNA sequence by affecting the affinity of histone with DNA. These modifications show a similar impact on the regulation of gene expression with DNA genetic codes, and thus are referred to as the “histone code”. Methylation of histone refers to methylation occurring at arginine or lysine residues at the N-terminal of H3 and H4 histones, which is mediated by histone methyltransferase. Methylation of lysine of histone has become an important regulatory mechanism of transcription and plays an important role in the formation of heterochromatin, inactivation of X chromosome, genomic imprinting, repair of DNA damage, and regulation of gene transcription [6-10]. The triple-methylation of lysine of Histone H3 at position 4 (H3K4me3) is generally associated with the activation of promoter [11], while the triple-methylation of lysine of Histone H3 at position 27 (H3K27me3) is generally associated with the silencing of the expression of promoter [12, 13]. In gene promoter region, the coexistence of the two histone modification states, H3K4me3 and H3K27me3, are referred as bivalent modification. Such a bivalent modification maintains the expression of the gene at a relatively low level and maintains the gene in a state of “being ready for transcription”. Such a state allows the gene to make a rapid response to appropriate stimulations (such as activation or inhibition of transcription and the like) [14-17].
Recently, growing numbers of studies pay attention to the role of methylation of lysine of histone played in embryonic development. Studies in zebrafish found that the genome becomes inactivated after fertilization and re-initiates transcription after maternal-zygotic transition [18-20]. The analytical results of triple-methylation of lysine in genomic histone H3 show that neither histone H3K27me3 inhibitory modification or H3K4me3 activating modification are detectable before transition. 80% genes show H3K4me3 modification after the transition is accomplished and the genome is activated, wherein some un-activated genes related to regulation of development also have H3K27me3 modification. These results indicate that bivalent or monovalent modification profile of chromatin histone H3 established during maternal-zygotic transition is likely to be associated with the establishment of totipotency [21]. Previous studies found that in mouse ESCs, co-localization of H3K4me3 and H3K27me3 is located within highly conserved regions consisting of about 2.5% genome, suggesting that such a bivalent modification state plays an important role in maintaining a state of “being ready for” activation in stem cells [14]. Studies on histone modifications in human ESCs found that, around the promoter, H3K4me3 modification widely spreads, while H3K27me3 is present only within 10% of gene promoter regions. In addition, the regions having H3K27me3 modification are also modified by H3K4me3 simultaneously. These genes modified by bivalent modification will be preferentially activated during ESC differentiation, suggesting that the existence of bivalent modification may be essential to maintain the development-related genes in a balanced state and to prepare for the future activation [15]; while those genes without any modifications will be in a state of suppression, and are completely silenced. A database established with ChIP-Seq detection results is used to analyze H3K4me3 and H3K27me3 modifications in multipotent neural the progenitor cells (NPCs), murine embryonic fibroblasts (MEFs) and primary human T cells. In NPCs or MEFs, there is a decline in the number of bivalently modified genes [10, 17]. This suggests that most of bivalently modified regions are specific to ESCs [14]. However, recent analysis in methylation profile of genome-wide histone shows that such bivalently modified regions are found in differentiated cells (such as T cells and MEFs). Thus, bivalent modification is not specific to ESCs [10 17, 22]. Histones of genes without any modification previously are re-modified by methylation, although there is a decline in the number of bivalently modified genes in human T cells when compared with ESCs, suggesting that such changes of the modification of histone may be associated with the specialization of T-cell and the inhibition of other pedigrees [15].
Although studies suggest that methylation of histone plays an important role in heterochromatin formation, inactivation of X-chromosome, genomic imprinting, repair of DNA damage and regulation of gene transcription, that methylation sites of histone are highly conserved among different species, and cells with different differentiation potentials have different profiles of methylation modification of histone, so far, people know little of the role and significance of methylation modification of histone in cell differentiation. Epigenetic regulation is a dynamic process, which makes epigenetic research complicated. In recent years, along with the rapid development of sequencing technology and the decrease in cost, the technology (ChIP-Seq) combining chromatin co-immunoprecipitation with sequencing has a wide range of applications [23, 24].
Stem cell transplantation can be used for the treatment of Parkinson's disease, cardiomyopathy, liver disease, for the induction of osteogenesis for the treatment of bone defects, and skin materials needed in the treatment of extensive burns, and the like. ASCs have advantages such as autologous, showing no signs of immunologic rejection during transplantation of tissue differentiated therefrom, and have a wide range of induced tissue types differentiated therefrom. Thus, ASCs show broad application prospects and are hopeful to become the main force in the stem cell transplantation treatment of various terminal stage organ diseases in the future. However, there are safety issues in stem cell transplantation. For example, it is reported that ESCs transplanted into the heart for the treatment of coronary heart disease may result in teratoma; the application of skeletal muscle stem cells may result in a rick of malignant arrhythmias; and bone marrow cells after being transplanted show severe myocardial calcification. Therefore, the successful treatment of stem cell transplantation depends on two important factors: (1) obtaining, purifying and amplifying seed stem cells in vitro; and (2) providing specific and functional differentiation of stem cells, in accordance with therapeutic purposes, in vivo. It is vital for stem cell transplantation treatment to not only control proliferation so as to avoid tumorigenesis but also initiate desired pathways to differentiate at a proper time. However, to address the series of issues, such as the acquisition of stem cells with appropriate potential of differentiation, identification according to therapeutic purposes, and transplantation into a body to provide specific differentiation at a proper time without the occurrence of teratoma, a set of indicators is needed to accurately identify and evaluate the differentiation potential, differentiation stage of stem cells and whether or not the stem cells can differentiate in a controllable and specific fashion and the like. Therefore, the prospect of pluripotent stem cells in clinical applications depend on their differentiation potential. The existing methods for examining the differentiation potential of stem cells derived from certain tissue are performed mainly by inducing differentiation and observing whether the stem cells can differentiate towards as many triploblastic pedigrees as possible. Such methods are time-consuming and require a lot of manpower and resources.
Chromatin Immunoprecipitation (ChIP) is the most important way for detecting histone modifications. ChIP is also known as binding site analysis, which is a powerful tool to study the in vivo interactions between proteins and DNA, and is usually used to study the binding site of transcription factors or specific modification sites of histone. ChIP is a method developed based on in vivo analysis. Its basic principle is to fix protein-DNA complex under living cell state and randomly cut the complex into small chromatin fragments within a range of certain lengths, and then precipitate the complex by using an immunology method, specifically enrich DNA fragments bound by target protein, and obtain information about protein-DNA interaction by the purification and detection of target fragments. Target fragments can be detected by tiling array or high-throughput sequencing, wherein the former is known as ChIP-on-chip while the latter is known as ChIP-Seq. The combination of ChIP and second-generation sequencing technology, ChIP-Seq technology, is able to highly efficiently detect the DNA segments interacting with histone or transcription factor and the like on a genome-wide scale.
The principle of ChIP-Seq is as follows: specifically enrich DNA fragments bound by target protein with ChIP; purify the fragments and establish a library; and subject the enriched DNA fragments to high-throughput sequencing. A researcher can obtain information about DNA segments interacting with histone or transcription factor and the like on a genome-wide scale by precisely locating millions of sequence tags in the genome.
ChIP-Seq data is the result of DNA sequencing, and provides researchers with resources for the further exploration of biological information. Researchers can carry out their studies in the following areas:
(1) Determine what kind of histone modification will exist at certain site of DNA strand;
(2) Detect the precise location of the binding site of RNA polymerase II and other trans-factors within the genome;
(3) Study the relationship between histone covalent modification and gene expression;
(4) Research CTCF transcription factors.
Therefore, the most basic material and indicators for broad clinical applications of stem cells can be provided if stem cells having appropriate differentiation potential can be obtained by using specific isolation, induction and screening systems. Genome-wide histone methylation profiles of stem cells at multiple grades can be studied by utilizing the role of “histone methylation code” in the prediction of cell differentiation and by using the combination of ChIP detection and bioinformatics (ChIP-Seq). The relationship between histone methylation modification and differentiation potential of stem cells can be found, the unified phenotype, culture condition and method for identification can be established for ASCs, and a set of indicators can be established in order to accurately identify and evaluate the differentiation potential, differentiation stage of stem cells, and determine whether or not stem cells can differentiate in a controllable and specific fashion. FIG. 7 is the technical schematic diagram of present invention.